Macrophage migration inhibitory factor gene polymorphisms in inflammatory bowel disease: an association study in New Zealand Caucasians and meta-analysis.

AIM: To investigate the association of macrophage migration inhibitory factor (MIF) promoter polymorphisms with inflammatory bowel disease (IBD) risk. METHODS: One thousand and six New Zealand Caucasian cases and 540 Caucasian controls were genotyped for the MIF SNP -173G > C (rs755622) and the repeat polymorphism CATT5₋8 (rs5844572) using a pre-designed TaqMan SNP assay and capillary electrophoresis, respectively. Data were analysed for single site and haplotype association with IBD risk and phenotype. Meta-analysis was employed, to assess cumulative evidence of association of MIF -173G > C with IBD. All published genotype data for MIF -173G > C in IBD were identified using PubMed and subsequently searching the references of all PubMed-identified studies. Imputed genotypes for MIF -173G > C were generated from the Wellcome Trust Case Control Consortium (and National Institute of Diabetes and Digestive and Kidney Diseases). Separate meta-analyses were performed on Caucasian Crohn's disease (CD) (3863 patients, 6031 controls), Caucasian ulcerative colitis (UC) (1260 patients, 1987 controls), and East Asian UC (416 patients and 789 controls) datasets using the Mantel-Haenszel method. The New Zealand dataset had 93% power, and the meta-analyses had 100% power to detect an effect size of OR = 1.40 at α = 0.05, respectively. RESULTS: In our New Zealand dataset, single-site analysis found no evidence of association of MIF polymorphisms with overall risk of CD, UC, and IBD or disease phenotype (all P values > 0.05). Haplotype analysis found the CATT5/-173C haplotype occurred at a higher frequency in New Zealand controls compared to IBD patients (0.6 vs 0.01; P = 0.03, OR = 0.22; 95%CI: 0.05-0.99), but this association did not survive bonferroni correction. Meta-analysis of our New Zealand MIF -173G > C data with data from seven additional Caucasian datasets using a random effects model found no association of MIF polymorphisms with CD, UC, or overall IBD. Similarly, meta-analysis of all published MIF -173G > C data from East Asian datasets (416 UC patients, 789 controls) found no association of this promoter polymorphism with UC. CONCLUSION: We found no evidence of association of MIF promoter polymorphisms with IBD.

Vitamin D receptor polymorphisms in colorectal cancer in New Zealand: an association study.

AIM: Polymorphisms of the vitamin D receptor (VDR) gene may be a risk factor for colorectal cancer (CRC). We investigated the association of three single nucleotide polymorphisms (SNPs) of the VDR gene with CRC in age and gender matched patients and controls of European origin in New Zealand. METHOD: CRC (N=200) and healthy control (N=200) samples were genotyped for the Fok1 (rs2228570), Taq1 (rs731236) and Cdx2 (rs11568820) polymorphisms using Taqman® SNP genotyping assays. Chi-squared analysis was used to test for overall association of VDR genotype with disease, and by age and gender subgroups. RESULTS: There were no significant associations of the three VDR SNPs with disease either by allelic frequencies (p=0.43-0.73) or genotypic distribution (p=0.15-0.90). Furthermore, no significant differences for allelic frequencies of the three SNPs were revealed in subgroup analysis by age (above/below median age of 72 yrs; p=0.38-0.91), gender (p=0.22-0.88), or age/gender (p=0.33-0.93) CONCLUSION: We found no evidence to suggest that the VDR SNPs Fok1, Taq1 and Cdx2 influence CRC risk in New Zealand Europeans.

NOD2 and ATG16L1 polymorphisms affect monocyte responses in Crohn's disease.

AIM: To assess whether polymorphisms in NOD2 and ATG16L1 affect cytokine responses and mycobacterium avium subspecies paratuberculosis (MAP) survival in monocytes from Crohn's disease (CD) patients. METHODS: Monocytes were isolated from peripheral blood of CD patients of known genotype for common single nucleotide polymorphisms of NOD2 and ATG16L1. Monocytes were challenged with MAP and bacterial persistence assessed at subsequent time-points. Cytokine responses were assayed using a Milliplex multi-analyte profiling assay for 13 cytokines. RESULTS: Monocytes heterozygous for a NOD2 polymorphism (R702W, P268S, or 1007fs) were more permissive for growth of MAP (P = 0.045) than those without. There was no effect of NOD2 genotype on subsequent cytokine expression. The T300A polymorphism of ATG16L1 did not affect growth of MAP in our model (P = 0.175), but did increase expression of cytokines interleukin (IL)-10 (P = 0.047) and IL-6 (P = 0.019). CONCLUSION: CD-associated polymorphisms affected the elimination of MAP from ex vivo monocytes (NOD2), or expression of certain cytokines (ATG16L1), implying independent but contributory roles in the pathogenesis of CD.

SLC11A1 polymorphisms in inflammatory bowel disease and Mycobacterium avium subspecies paratuberculosis status.

AIM: To test for association of SLC11A1 with inflammatory bowel disease (IBD) and Mycobacterium avium subspecies paratuberculosis (MAP) status in a Caucasian cohort. METHODS: five hundred and seven Crohn's disease (CD) patients, 474 ulcerative colitis (UC) patients, and 569 healthy controls were genotyped for SLC11A1 1730G>A and SLC11A1 469+14G>C using pre-designed TaqMan SNP assays. χ(2) tests were applied to test for association of single nucleotide polymorphisms (SNPs) with disease, and the presence of MAP DNA. RESULTS: SLC11A1 1730G>A and SLC11A 1469+14G>C were not associated with CD, UC, or IBD. The SLC11A1 1730A minor allele was over-represented in patients who did not require immunomodulator therapy (P = 0.002, OR: 0.29, 95% CI: 0.13-0.66). The frequency of the SLC11A1 469+14C allele was higher in the subset of study participants who tested positive for MAP DNA (P = 0.02, OR: 1.56, 95% CI: 1.06-2.29). No association of SLC11A1 1730G>A with MAP was observed. CONCLUSION: Although SLC11A1 was not associated with IBD, association with MAP suggests that SLC11A1 is important in determining susceptibility to bacteria implicated in the etiology of CD.

Association of higher DEFB4 genomic copy number with Crohn's disease.

OBJECTIVES: Human beta-defensin 2 (hBD-2 or DEFB4) is a highly inducible, antimicrobial peptide, which may have an important role in the innate immune response at epithelial surfaces. Genomic copy number of DEFB4 is polymorphic, with most individuals possessing 3-5 copies. Increased DEFB4 copy number is a susceptibility factor for psoriasis, whereas a single study in a Crohn's disease (CD) cohort reported that decreased DEFB4 copy number is associated with colonic inflammation. Here, we analyze association of DEFB4 copy number with CD in a New Zealand case-control cohort of European origin. METHODS: DEFB4 gene copy number was determined using TaqMan quantitative PCR in 466 CD patients and 329 controls. DNA samples, independently genotyped for DEFB4 copy number by alternative methods, were used to validate the assay. RESULTS: Increased DEFB4 genomic copy number was seen in CD patients compared with controls. Individuals with >4 copies had a significantly higher risk of developing CD than those with <4 copies (odds ratio 1.54; 95% confidence interval 1.13-2.09, P=5e-05). DEFB4 genomic copy number did not differ by disease location within the CD cohort (P=0.948), nor did analysis of CD patients who had undergone surgery detect association of decreased DEFB4 genomic copy number (<4) in colonic CD compared with ileal CD (P=0.120). CONCLUSIONS: Our results indicate that elevated DEFB4 copy number is a risk factor for CD (irrespective of intestinal location), and challenge previous data supporting positive association of lower DEFB4 genomic copy number with colonic CD.

Incidence of Mycobacterium avium subspecies paratuberculosis in a population-based cohort of patients with Crohn's disease and control subjects.

OBJECTIVE: To define the incidence of Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease (CD) and in control subjects. METHODS: Blood samples from 361 CD patients from a previously described population-based inflammatory bowel disease (IBD) cohort and 200 blood donor controls, of known NOD2 genotype, were screened by PCR for MAP-specific IS900 DNA. These results were correlated with NOD2 genotype. RESULTS: The PCR assay was capable of detecting 20 fg of purified MAP DNA, equivalent to roughly 100 MAP cells/mL of blood. MAP-specific IS900 DNA was detected in 33.8% of CD cases and 21.5% of controls (OR 1.86, 95% CI 1.247-2.785, P= 0.002). All study participants were genotyped for the NOD2 mutations 2104C>T (R702W), 2722G>C (G908R), and 3020insC (1007fs). Carriage of one or two NOD2 mutations was not associated with a significantly higher risk of CD (OR 0.75, 95% CI 0.465-1.207, P= 0.234). No significant association was seen in the CD cohort for carriage of one or two NOD2 mutations and MAP status (OR 0.883, 95% CI 0.494-1.579, P= 0.675). CONCLUSIONS: Screening peripheral blood using IS900 PCR indicated that MAP DNA could be detected in a significant proportion of CD cases from a large population-based cohort, and also, in control subjects. The over-representation of MAP DNA in CD suggests either a role or a probable role for MAP in the etiology of CD.